Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure 1 - Lethal mutation of either Mcm2 or Mcm3 NLSs can be rescued by fusing tandem SV40 NLSs to a single Mcm protein in the Mcm2-7 complex.

  A. The URA3 marked plasmid pKI1500 (mcm2-nls-GFP) is integrated in the following strains by homologous recombination 5’ of the MCM2 ORF: YJL1231 (MCM2::{mcm2-nls-GFP, URA3}), YJL3074 (MCM2::{mcm2-nls-GFP, URA3} SVNLS₂-MCM3), YJL3070 (MCM2::{mcm2-nls-GFP, URA3} MCM4-SVNLS₂), YJL3072 (MCM2::{mcm2-nls-GFP, URA3} MCM5-SVNL S₂), and YJL3076 (MCM2::{mcm2-nls, URA3} MCM6-SVNL S₂). pKI1495 (mcm2-nls-GFP-SVNL S₂) is similarly integrated in YJL1228 (MCM2::{SVNL S₂-mcm2-nls, URA3}). These strains contain both a WT and mutant copy of the MCM2 locus in direct repeat, and homologous recombination between these loci would result in excision of the integrated plasmid. The strains were plated on 5-FOA to select for this excision, which depending on the position of the recombination event, would leave a single MCM2 locus containing either the WT or mutant MCM2 NLS sequence (see diagram). PCR and restriction analysis was used to distinguish between these two outcomes. The number of excisants containing the mutation and the total number of excisants screened are tabulated.

  B. The URA3 marked plasmid pKI1362 (GFP-mcm3-nls) is integrated by plasmid integrated by homologous recombination 3’ of the MCM3 ORF in the following strains: YJL2675 (MCM3::{GFP-mcm3-nls, URA3}), YJL3445 (MCM3::{GFP-mcm3-nls, URA3} MCM2-SVNL S₂), YJL3438 (MCM3::{GFP-mcm3-nls, URA3} MCM4-SVNL S₂), YJL3441 (MCM3::{GFP-mcm3-nls, URA3} MCM5-SVNL S₂), and YJL3444 (MCM3::{GFP-mcm3-nls, URA3} MCM6-SVNL S₂). pKI1356 (SVNL S₂-GFP-mcm3-nls) is similarly integrated in YJL2665 (MCM3::{SVNL S₂-GFP-mcm3-nls, URA3}). These strains were plated on 5-FOA to select for plasmid excision and the excisants were analyzed as described in Supplementary Figure 1A.

• Supplementary Figure 2 - The phosphomimic mutations of the Mcm3 CDK consensus sites can slow or arrest the cell cycle and induce an accumulation of G2/M cells.

  A. YJL1265 (GFP-mcm3-cdk5ED) and YJL2160 (GFP-MCM3) cells growing exponentially in YEPD medium were sonicated and plated on YEPD plates at 23 C. The number of cell lobes in each of 200 colonies was counted at the indicated times. B. YJL1265 (GFP-mcm3-cdk5ED) and YJL2160 (GFP-MCM3) cells growing exponentially in YEPD medium were fixed in ethanol for FACS analysis and in formaldehyde for budding indices.

• Supplementary Table 1 - Supplementary Table 1. Base position is listed relative to the first nucleotide of the ORF (+1) with starting nucleotide on left and substituted nucleotide on right. In most cases the starting nucleotide is wild type (upper case) and substituted nucleotide is mutant (lower cases), but in a few cases a mutated nucleotide is reverted back to wild-type. Base subsitution for all alleles aside from the silent allele were introduced into the silent allele.
• Supplementary Table 2
• Supplementary Table 3
• Supplementary Table 4
• Supplementary Material